Human GABAB receptor 1 promoters

ABSTRACT

The present invention relates to nucleic acid molecules constituting GABA B  receptor 1 promoters P1a and/or P1b, and to methods for screening for compounds which are modulators of GABA B  receptor 1 transcription, said methods comprising the use of nucleic acid molecules constituting GABA B  receptor P1a and/or P1b promoters.

This application is a divisional of U.S. application Ser. No.09/622,745, filed Aug. 22, 2000, now U.S. Pat. No. 6,933,124, which isthe National Stage of International Application No. PCT/SE00/00878,filed May 4, 2000.

FIELD OF THE INVENTION

The present invention relates to nucleic acid molecules constitutingGABA_(B) receptor 1 promoters P1a and/or P1b, and to methods forscreening for compounds which are modulators of GABA_(B) receptor 1transcription, said methods comprising the use of nucleic acid moleculesconstituting GABA_(B) receptor P1a and/or P1b promoters.

BACKGROUND

GABA_(B) Receptor 1

GABA (γ-aminobutyric acid) is an endogenous neurotransmitter in thecentral and peripheral nervous systems. Receptors for GABA havetraditionally been divided into GABA_(A) and GABA_(B) receptor subtypes.GABA_(B) receptors (for a review see Kerr, D.I.B. and Ong, J. (1995)Pharmac. Ther. vol. 67, pp. 187-246) belong to the superfamily ofG-protein coupled receptors. GABA_(B) receptor agonists are described asbeing of use in the treatment of CNS disorders, such as musclerelaxation in spinal spasticity, cardiovascular disorders, asthma, gutmotility disorders such as irritable bowel syndrome and as prokineticand anti-tussive agents. GABA_(B) receptor agonists have also beendisclosed as useful in the treatment of emesis (WO 96/11680) and refluxdisease (WO 98/11885).

The cloning of the cDNA encoding the rat GABA_(B) receptors spliceisoforms GABA_(B)R1a and GABA_(B)R1b is disclosed by Kaupmann et al.(1997) Nature, vol. 386, 239-246. The mature rat GABA_(B)R1b differedfrom GABA_(B)R1a in that the N-terminal 147 residues were replaced by 18different residues. It was presumed that the rat GABA_(B)R1a and -breceptor variants are derived from the same gene by alternativesplicing.

The cloning of the cDNA encoding the human GABA_(B) receptor GABA_(B)R1bis disclosed in WO 97/46675.

The cloning of the human GABA_(B) receptor 1 gene and the elucidation ofthe exon-intron organization is in part or fully disclosed inPCT/SE98/01947, in EMBL HS271M21 (GenBank AL031983), EMBL AJ010170 toAJ010191, in Peters, H C et al., Neurogenetics 2; 47-54 (1998) and inGoei, V L et al. Biological Psychiatry. 44; 659-66 (1998). The humanGABA_(B) receptor 1 gene consists of 23 exons, spanning over a distanceof 30 kb (FIG. 1). The elucidation of the gene organization revealedthat the human GABA_(B)R1a and GABA_(B)R1b are splice variants encodedby a single gene. The GABA_(B)R1a and GABA_(B)R1b isoforms aredifferentially expressed, at least in the rat (Kaupmann et al. (1997)Nature, vol. 386, 239-246). The physiological consequences of multipleGABA_(B) receptor 1 splice isoforms has not yet been determined, buttheir existence constitute an opportunity for the development ofspecific pharmaceutical agents.

GABA_(B) Receptor 2

Based on its homology with the mammalian GABA_(B)R1 cDNA, a secondmember of the GABA_(B) receptor family was identified (Jones, K A etal., Nature 396; 674-679 (1998), White, J A et al., Nature 396; 679-682(1998), Kaupmann, K et al., Nature 396; 683-687 (1998), WO 99/20751).The corresponding protein, GABA_(B)R2, forms heteromers with GABA_(B)R1aand R1b, resulting in cell surface expression of a functional GABA Breceptor (Kuner, R et al. Science 283, 74-77 (1999)). At least inrecombinant expression systems, GABA_(B)R1 and R2 coexpression isnecessary for the formation of a functional GABA_(B) receptor. Jones etal. (Nature 396; 674-679 (1998)) disclosed that a GABA_(B)R1:GABA_(B)R2stochiometry of 1:1 is an optimal ratio for successful cell surfaceexpression of a ligand binding and functional GABA_(B) receptor. Thus,modulating GABA_(B)R1 expression could alter the stochiometry betweenGABA_(B)R1 and other interacting proteins and be a means to regulatesignaling through GABA_(B) receptors and thereby interfere with variousphysiological processes.

Transcriptional Regulation

Gene regulation is mediated by specific DNA elements in the promoterthat directs binding of transcription factors, which thereby mediatetranscription of the gene. Eukaryotic transcription factors can bedivided in two main groups i) basal transcription factors that interactwith promoter sequences proximal to the start of transcription, therebyinitiating transcription upon recruitment of RNA polymerase II and ii)transcription factors that bind to specific distal promoter elements,thereby modulating the transcription upon contact with the basaltranscription machinery. The DNA sequence that directs the start oftranscription in most eukaryotic genes is the TATA-box, which is oftenlocated approximately 30 base pairs upstream from the start oftranscription. However, the TATA-box is not a prerequisite forinitiation of transcription as there are many promoters, including theGABA_(B)R1 promoters described in this study, that lack a TATA-box. Afundamental physiological process in the eukaryotic organism is thatcells can communicate with their environment and respond toextracellular stimuli through signaling molecules, such as hormones andgrowth factors. The final event for such signaling is the binding oftranscription factors to specific distal promoter elements leading tofor example up-regulated or tissue specific gene expression. Because oftheir regulatory role, signaling molecules are putative targets forscreening of therapeutic agents. The presence of two distinct anddifferentially regulated promoters within the human GABA_(B) receptor 1gene, disclosed in this patent application, makes it possible to screenfor therapeutic agents selectively regulating expression of GABA_(B)receptor 1a and 1b-type splice isoforms.

Indications

Compounds which are modulators of GABA_(B) receptor 1 transcription arepotentially useful in the treatment of disorders which are related toneurally-controlled physiological responses regulated by GABA_(B)receptors, e.g. CNS disorders such as muscle relaxation in spinalspasticity, Alzheimer's disease and other dementias, psychiatric andneurological disorders such as depression, anxiety and epilepsy,cardiovascular disorders, asthma, gut motility disorders such asirritable bowel syndrome, emesis and reflux disease. In some humans, thelower esophageal sphincter (LES) is prone to relaxing more frequentlythan in other humans. As a consequence, fluid from the stomach can passinto the esophagus since the mechanical barrier is temporarily lost atsuch times, an event hereinafter referred to as “reflux”.

Gastro-esophageal reflux disease (GERD) is the most prevalent uppergastrointestinal tract disease. Current therapy has aimed at reducinggastric acid secretion, or by reducing esophageal acid exposure byenhancing esophageal clearance, lower esophageal sphincter tone andgastric emptying. The major mechanism behind reflux has been consideredto depend on a hypotonic lower esophageal sphincter. However, recentresearch (e.g. Holloway & Dent (1990) Gastroenterol. Clin. N. Amer. 19,517-535) has shown that most reflux episodes occur during transientlower esophageal sphincter relaxations (TLESR), i.e. relaxations nottriggered by swallows. It has also been shown that gastric acidsecretion usually is normal in patients with GERD. Consequently, thereis a need for compounds which reduce the incidence of TLESR and therebyprevent reflux.

DESCRIPTION OF THE INVENTION

This invention relates to nucleic acid molecules constituting GABA_(B)receptor 1 promoters and fragments of said promoters. By GABA_(B)receptor 1 promoters is understood the nucleic acids sequences upstreamof the ATG translation initiation codon of GABA_(B) receptor 1a of theGABA_(B) receptor 1 gene, designated P1a, and the nucleic acidssequences upstream of the ATG translation initiation codon of GABA_(B)recpetor 1b of the GABA_(B) receptor 1 gene, designated P1b, asillustrated in FIG. 1.

In the present context the term “promoter” is meant to include corepromoter sequences proximal to the start of transcription and upstreampromoter elements which bind constitutively active transcriptionfactors, as well as distal promoter elements which bind specifictranscription factors.

Accordingly, the present invention provides a nucleic acid moleculeconstituting a human GABA_(B) receptor 1 promoter P1a, or a functionallyequivalent modified form thereof, or active fragments thereof. Thepresent invention also provides a nucleic acid molecule constituting ahuman GABA_(B) receptor 1 promoter P1b, or a functionally equivalentmodified form thereof, or active fragments thereof. By a functionallyequivalent modified form is understood nucleic acids modified from theoriginal sequence that can bind transcription factors. By activefragments of the promoters is understood nucleic acid fragments that canbind transcription factors.

In preferred forms of the invention the said nucleic acid molecule isselected from:

-   (a) a nucleic acid molecule comprising a nucleotide sequence set    forth as SEQ ID NO: 1;-   (b) a nucleic acid molecule comprising a nucleotide sequence capable    of hybridizing, under stringent conditions, to a nucleotide sequence    complementary to the DNA molecule as defined in (a).

In other preferred forms of the invention the said nucleic acid moleculeis selected from:

-   (a) a nucleic acid molecule comprising a nucleotide sequence set    forth as SEQ ID NO: 2;-   (b) a nucleic acid molecule comprising a nucleotide sequence capable    of hybridizing, under stringent conditions, to a nucleotide sequence    complementary to the DNA molecule as defined in (a).

In another preferred form of the invention the said nucleic acidmolecule may be a nucleic acid molecule constituting a human GABA_(B)receptor 1 promoter P1a, or a functionally equivalent modified formthereof, or active fragments thereof, in combination with a nucleic acidmolecule constituting a human GABA_(B) receptor 1 promoter P1b, or afunctionally equivalent modified form thereof, or active fragmentsthereof.

In another preferred form of the invention the said nucleic acidmolecule may be a nucleic acid molecule selected from:

-   (a) a nucleic acid molecule comprising a nucleotide sequence set    forth as SEQ ID NO: 1;-   (b) a nucleic acid molecule comprising a nucleotide sequence capable    of hybridizing, under stringent conditions, to a nucleotide sequence    complementary to the DNA molecule as defined in (a);    in combination with a nucleic acid molecule is selected from:-   (a) a nucleic acid molecule comprising a nucleotide sequence set    forth as SEQ ID NO: 2;-   (b) a nucleic acid molecule comprising a nucleotide sequence capable    of hybridizing, under stringent conditions, to a nucleotide sequence    complementary to the DNA molecule as defined in (a).

It should thus be understood that the nucleic acid molecules accordingto the invention is not to be limited strictly to molecules comprisingthe sequences set forth as SEQ ID: 1 and 2. Rather the inventionencompasses nucleic acid molecules carrying modifications likesubstitutions, small deletions, insertions or inversions, whichnevertheless have substantially the biochemical activity of the GABA_(B)receptor promoters 1a and/or 1b according to the invention. Included inthe invention are consequently nucleic acid molecules, the nucleotidesequence of which is at least 95% homologous, preferably at least 96%,97%, 98% or 99% homologous, with the nucleotide sequence shown as SEQ IDNO: 1 or 2 in the Sequence Listing.

The term “stringent hybridization conditions” is known in the art fromstandard protocols (e.g. Current Protocols in Molecular Biology, editorsF. Ausubel et al., John Wiley and Sons, Inc. 1994) and could beunderstood as as stringent or more stringent than those defined by e.g.hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at +65° C., and washing in 0.1×SSC/0.1% SDS at+68° C.

It will be appreciated that the nucleic acid sequences shown in theSequence Listing is only an example within a large but definite group ofnucleic acid sequences, which will have the GABA_(B) receptor promoteractivity.

In yet another aspect, the invention provides a vector transformed witha nucleic acid molecule of the present invention. The said vector cane.g. be a replicable expression vector, which carries a nucleic acidmolecule according to the invention. In the present context the term“replicable” means that the vector is able to replicate in a given typeof host cell into which is has been introduced. Examples of vectors areviruses such as bacteriophages, cosmids, plasmids and otherrecombination vectors. Nucleic acid molecules are inserted into vectorgenomes by methods well known in the art.

Another embodiment of the present invention is an expression systemcomprising nucleic acid molecules encoding GABA_(B) receptor promotersP1a and/or P1b, or functionally equivalent modified forms, or activefragments thereof.

In preferred forms of this embodiment of the invention the said nucleicacid molecule is selected from: (a) a nucleic acid molecule comprising anucleotide sequence set forth as SEQ ID NO: 1 and/or SEQ ID NO: 2; (b) anucleic acid molecule comprising a nucleotide sequence capable ofhybridizing, under stringent conditions, to a nucleotide sequencecomplementary to the polypeptide coding region of a DNA molecule asdefined in (a).

In another preferred form of this embodiment of the invention the saidnucleic acid molecule may be a nucleic acid molecule constituting ahuman GABA_(B) receptor 1 promoter P1a, or a functionally equivalentmodified form thereof, or active fragments thereof, in combination witha nucleic acid molecule constituting a human GABA_(B) receptor 1promoter P1b, or a functionally equivalent modified form thereof, oractive fragments thereof.

In another preferred form of this embodiment of the invention the saidnucleic acid molecule may be a nucleic acid molecule selected from:

-   (a) a nucleic acid molecule comprising a nucleotide sequence set    forth as SEQ ID NO: 1;-   (b) a nucleic acid molecule comprising a nucleotide sequence capable    of hybridizing, under stringent conditions, to a nucleotide sequence    complementary to the DNA molecule as defined in (a);    in combination with a nucleic acid molecule is selected from:-   (a) a nucleic acid molecule comprising a nucleotide sequence set    forth as SEQ ID NO: 2;-   (b) a nucleic acid molecule comprising a nucleotide sequence capable    of hybridizing, under stringent conditions, to a nucleotide sequence    complementary to the DNA molecule as defined in (a).

The expression system may, in addition, comprise a reporter gene, thepromoter and the reporter gene being positioned so that the expressionof the reporter gene is regulated by the GABA_(B) receptor 1 promotersP1a and/or P1b. Suitable expression systems according to the inventionare e.g. bacterial or yeast plasmids, wide host range plasmids andvectors derived from combinations of plasmid and phage or virus DNA.Furthermore, an origin of replication and/or a dominant selection markercan be present in the vector according to the invention. Suitablereporter genes that can be used for the construction of expressionsystems according to the invention are e.g. the firefly luciferase gene,the bacterial chloramphenicol acetyl transferase (CAT) gene, theβ-galactosidase (β-GAL) gene, and the green fluorescent protein (GFP)gene.

A further aspect of this embodiment of the invention is a host celltransfected with an expression system comprising nucleic acid moleculesconstituting GABA_(B) receptor promoters P1a and/or P1b, or functionallyequivalent modified forms thereof, or active fragments thereof.

Suitable host cells are cells known to express GABA_(B) receptors orcells known to express transcription factors, which can influence thetranscription of GABA_(B) receptors. Host cells transfected with DNAencoding specific transcription factors can preferably be used to studythe interaction with defined transcription factors and the GABA_(B)receptor promoter.

Another embodiment of the invention is a method for the assay ofGABA_(B) receptor promoter activity said method comprising the use of ahost cell transfected with an expression system comprising nucleic acidmolecules constituting GABA_(B) receptor promoters P1a and/or P1b, orfunctionally equivalent modified forms thereof, or active fragmentsthereof.

A further embodiment of the present invention is a method for thescreening of compounds which are modulators of GABA_(B) receptor 1transcription, said method comprising the use of nucleic acid moleculesconstituting GABA_(B) receptor P1a and/or P1b promoters.

Accordingly, the present invention provides a method for screeningcompounds which are modulators of GABA_(B) receptor 1 transcription,comprising the steps of: (a) transfecting a cell host with a suitableexpression system comprising a nucleic acid molecule constituting humanGABA_(B) receptor 1 promoter P1A, and/or a human GABA_(B) receptor 1promoter P1B or functionally equivalent modified forms, or activefragments thereof coupled to a reporter gene; (b) contacting a testcompound with the cell; and (c) determining whether the test compoundmodulates the level of expression of the reporter gene.

In one aspect of this embodiment of the invention it is provided amethod of screening of compounds which are modulators of GABA_(B)receptor 1 transcription, wherein the cell host endogenously expressesGABA_(B) receptor 1.

In another aspect of this embodiment of the invention it is provided amethod of screening of compounds which are modulators of GABA_(B)receptor 1 transcription, wherein the said cell host is furthertransfected with a suitable expression system comprising a nucleic acidmolecule encoding one or more specific transcription factors.Preferably, the said transcription factors are selected from the group:CREB-1, CREB-2, CREM-1, ATF-1, ATF-2, ATF-3, ATF-4, Sp1, Sp2, Sp3, Sp4,AP-1, and AP-2.

A further embodiment of the invention is a transgenic non-human animalwhose genome comprises an expression system comprising nucleic acidmolecules constituting GABA_(B) receptor promoters P1a and/or P1b, orfunctionally equivalent modified forms thereof, or active fragmentsthereof, coupled to a reporter gene.

Such transgenic non-human mammals can be generated by insertion of DNAcomprising GABA_(B) receptor promoters by microinjection, retroviralinfection or other means well known to those skilled in the art, intoappropriate fertilized embryos to produce a transgenic animal (Hogan B.et al Manipulating the Mouse Embryo, A Laboratory Manual, Cold SpringHarbor Laboratory (1986)).

Accordingly, the present invention provides a method for the screeningof compounds which are modulators of GABA_(B) receptor 1 transcription,comprising the use of a transgenic non-human animal whose genomecomprises an expression system comprising nucleic acid moleculesconstituting GABA_(B) receptor promoters P1a and/or P1b, or functionallyequivalent modified forms thereof, or active fragments thereof, coupledto a reporter gene, or tissues or cells isolated from such transgenicanimals.

EXAMPLES

The following examples are preferred non-limiting examples embodyingpreferred aspects of the invention.

Example 1 Isolation and Identification of Human GABA_(B)R1 Promoters P1aand P1b

Genomic DNA containing the human GABA_(B) receptor gene was isolatedfrom human genomic libraries and genomic DNA. Human genomic librarieswere obtained from Clontech (Palo Alto, Calif., USA). The libraries wereconstructed from female leukocyte DNA (catalogue # HL1111J), cloned intoλEMBL-3 vector. The average size of inserts are 16 kb and the number ofindependent clones are 1.7×10⁶. Human genomic DNA was obtained fromClontech (catalogue # 6550-1). In order to isolate recombinant phasescontaining exon and intron sequences of the human GABA_(B) receptorgene, 48 individual bacterial plates with a diameter of 150 mm andapproximately 4×10⁴ individual plaques per plate, were screened. Themethods and solutions used were as described in the Library ProtocolHandbook: General Procedures for the Hybridization of Lambda PhageLibraries w/DNA Probes (Clontech) with some modifications as will beapparent from the following.

The experiment was carried out essentially as follows. The numbers aregiven per plate basis. A sample of the phage library diluted in 0.1 mlsterile lambda diluent was prepared in order to obtain an estimatedtiter of 40,000 pfu (plaque forming units). A 0.6 ml LB-medium cultureof the E. coli host strain K802 (obtained from Clontech) was infectedwith 40000 pfu recombinant phages for 15 minutes at 37° C. The culturewas then mixed with 7 ml top agarose (6.5 g of agarose added per literLB) and poured onto LB plates. The plates were incubated at 37° C. forapproximately 7 hours. The plates were then chilled at +4° C.

Plaque hybridization experiments were as follows. Membrane filters,Colony/Plaque Screen (DuPont, Wilmington, Del., USA), were placed ontothe top of the plates for 3 minutes. For denaturation of DNA the filterswere removed and floated in 0.5 M NaOH on a plastic wrap for 2 minutes,with the plaque side up. This step was repeated once to ensure efficientdenaturation. Following neutralization the membrane filters were placedin 1M Tris-HCl pH 7.5, two times 2 minutes and allowed to dry.

To obtain probes for DNA hybridization screening of the membranefilters, a GABA_(B) receptor cDNA clone was digested with SacII and a479 bp fragment, separated by agarose electrophoresis, excised andtransferred to a polypropylene microcentrifuge tube. Additional probeswere obtained by PCR amplification of various regions of the GABA_(B)receptor cDNA. The isolated cDNA fragment was ³²P-labeled usingMegaprime DNA labeling system (Amersham Pharmacia Biotech, Uppsala,Sweden) by the following procedure. Water was added at a ratio of 3 mlper gram of gel, and placed in a boiling water bath for 7 minutes tomelt the gel and denature the DNA. A volume of DNA/agarose solutioncontaining 25 ng of DNA was added to the labeling reaction, according tothe supplier's instructions. Labeled nucleotides were removed from DNAlabeling reactions using MicroSpin™ G-50 Columns (Amersham PharmaciaBiotech, Uppsala, Sweden).

The DNA hybridization reaction was performed under stringent conditionsaccording to the method described below. The filter membranes wereprehybridized at 65° C. for at least 1 hour in a solution composed of 1%SDS, 1M NaCl, and 10% dextran sulfate using a hybridization oven (HybaidLtd, Ashford, UK). Following prehybridization a solution containingdenatured herring sperm DNA of a final concentration of 100 μg/ml andthe ³²P-labeled DNA probe at a concentration <10 ng/ml (for optimalsignal to background ratio) was added to the prehybridization solutionand the membrane filters were incubated at 65° C. for 10-20 hours.Following the removal of the hybridization solution the membrane filterswere first washed in a 2×SSC (0.3M NaCl, 0.03M Na-citrate), 1% SDSsolution two times for 5 minutes at room temperature. In the next step,the membrane filters were incubated at 60° C. two times for 30 minuteseach in the same solution. In a third step, the filters were washed twotimes at room temperature in 0.1×SSC. Finally, the membrane filters wereplaced on a sheet of filter paper with the DNA face up, and allowed todry. The dried membrane filters were then exposed to X-ray films andautoradiographed.

Of the approximately 2×10⁶ individual plaques analyzed, four hybridizingplaques were detected and isolated. These four isolates were designated#GR1, #GR12, #GR13 and #GR41, respectively. After several rescreeningexperiments, the recombinant phage DNA was purified using Qiagen LambdaMidi Kit (Qiagen GmbH, Germany). The purified DNA was digested with SalIand the fragments representing the inserts were isolated by agaroseelectrophoresis.

The 16 kb insert of isolate #GR 13 was cloned into SalI digestedlinearized pUC19, resulting in the plasmid pAM364. The insert wasanalyzed by PCR, restriction mapping and hybridization to ³²P-labeledDNA fragments representing various regions of the GABA_(B) receptorcDNA.

The cloned fragment in the plasmid pAM364 was characterized byrestriction enzyme mapping, using EcoRI, HindIII, PstI, and BamHI. Theapproximate positions of the exons and the approximate size of theintrons were analyzed and determined by PCR-based exon-exon linking andagarose gel electrophoresis.

In order to facilitate nucleotide sequence analysis, 7 restrictionsub-fragments derived from pAM364 were isolated and cloned individuallyinto pUC19. The following strategy was employed; by combining PCRprimers located within the pUC19 sequence either upstream or downstreamof the cloning site, with a PCR primer with defined orientation andspecific for the GABA_(B) receptor derived subcloned fragment allowedthe sequence determination.

The inserts were subjected to nucleotide sequence analysis. Thenucleotide sequences for all subclones were determined using a ThermoSequenase dye terminator cycle sequencing pre-mix kit (AmershamPharmacia Biotech, Uppsala, Sweden). As primers for sequencing reactionsspecific oligonucleotides complementary to pUC19 or primerscomplementary to the human GABA_(B) receptor cDNA were used.

The sequence of the human GABA_(B) receptor gene fragment cloned in theplasmid pAM364 has previously been revealed (see PCT/SE98/01947). Thisgenomic fragment was shown to contain the complete exons 1-11 and thecomplete introns 1-10 of the human GABA_(B) receptor gene as well as >3kb sequence upstream of exon 1. The elucidation of the gene organizationrevealed that the human GABA_(B)R1a and GABA_(B)R1b are splice variantsencoded by a single gene.

In order to localize the putative human GABA_(B) receptor promoter, weinvestigated the genomic sequence for the presence of consensussequences of known regulatory promoter elements. To our surprise, wefound that promoter elements, clustered in two regions: one regionupstream of exon 1, and the other region in intron 5, just upstream ofexon 6. We concluded that the human GABA_(B) receptor may be regulatedby two independent promoters, and not by one single promoter asexpected. The first putative promoter, denoted P1a (SEQ ID NO:1) anddescribed in detail below, may regulate transcription ofGABA_(B)R1a-type splice variants and the second putative promoter,denoted P1b (SEQ ID NO: 2) and described in detail below, may regulatetranscription of GABA_(B)R1b-type splice variants.

As indicated in the schematic representation of P1a and P1b (FIG. 2),both putative promoters lack a TATA box. However P1b has an initiator(Inr) element in position 4375-4381 which is located 24-30 bp upstreamof the position corresponding to the 5′ end of the longest known cDNAisolated with “rapid amplification of cDNA ends” (RACE) PCRamplification. The Inr element may therefore direct the start oftranscription from P1b. P1a contains neither a TATA or an Inr elementand the transcription from R1a may therefore initiate from differentstart sites which is often the case in promoters lacking TATA boxes orInr elements. Both P1a and P1b contain multiple GC rich regions at pos.3009-3016, 3037-3044 and 3116-3123 in R1a and pos. 4080-4087, 4196-4204,4241-4249 and 4272-4279 in R1b, which are potential binding site for theSP1 family of transcription factors. SP1 binding sites are often foundin TATA lacking promoters where they often substantially contribute totranscription. In addition, to the indicated GC sequences in FIG. 2there are also other GC motifs that may function as SP1 binding sites.P1a further contains an activator protein-1 (AP-1) site at position1497-1503. AP-1 sites are recognized by AP-1 transcription factors whichconsists of homodimers of members of the Jun family or Fos/Junheterodimeric complexes. AP-1 complexes also interact, byprotein-protein interactions, with members of the steroid receptorfamily and are therefore also targets for steroid receptor signaling. Inaddition to the GC motifs P1b also contain an activator protein-2 (AP-2)site at position 3844-3851 and a cAMP responsive element (CRE) atposition 4308-4315. Especially the finding of a consensus CRE (TGACGTCA)is interesting as this promoter element is found in many genes regulatedby cAMP which are bound and regulated by members of the ATF/CREB genefamily. This sequence may therefore be an important target for cAMPmediated signaling via G-protein coupled receptors, including GABA_(B)receptors.

We conclude that transcription of the human GABA_(B) receptor gene maybe regulated by two putative promoters, P1a and P1b, that mayindependently regulate expression of human GABA_(B) receptor 1a and 1bsplice isoforms, respectively.

Example 2 Determination of GABA_(B)R1 Promoter P1a and P1b Activity

To experimentally determine if P1a and P1b indeed act as promoters, wefused these regions to a cDNA encoding firefly luciferase to be used asa reporter of promoter activity in transfected cells.

Reporter constructs containing R1a and R1b promoter fragments weregenerated by PCR using plasmid pAM 364, containing genomic sequencecovering the promoter regions, as template. PCR reaction was performedby standard procedure (Perkin Elmer). Briefly, an initial denaturationat 95° C. for 4 min was followed by 35 cycles of denaturation at 95° C.for 1 min, annealing at 60° C. for 1 min, elongation at 72° C. for 1 minand finally a 7 min elongation at 72° C. In the sequence for primersused to generate promoter fragments (for details see Table 1. below), aNhe I and Hind III endonuclease restriction enzyme site was introduced,in 5′ and 3′ primers respectively, to enable sub-cloning into Nhe I/HindIII digested pGL3-Basic luciferase reporter vector (Promega). Hence,complete promoter-reporter constructs (pAM440, pAM438 and pAM436)contain R1a and R1b promoter fragments (indicated size see table) fusedto the luciferase reporter gene. Plasmid DNA were purified using Qiagentip-100 columns according to suppliers instruction. Correct fragmentinsertion was verified by DNA sequencing.

TABLE 1 Nucleotide sequence of primers used to generate promoterfragments Primer Restriction Promoter No. site sequence PositionSequence 5′-3′ 1582 HindIII SEQ ID 3440-3424 AAG CTT CTC GGC (AAG CTT)NO: 1 GCG CGG GCC CG (SEQ ID NO: 3) 1583 NheI SEQ ID 2341-2362 GCT AGCCAA GAG (GCT AGC) NO: 1 CTT CTG GAG CCG (SEQ ID NO: 4) 1584 NheI SEQ ID720-741 GCT AGC TGT TAC (GCT AGC) NO: 1 ATG CAG AGC AAT C (SEQ ID NO: 5)1585 HindIII SEQ ID 4439-4421 AAG CTT CCT ACG (AAG CTT) NO: 2 GCC CCCGCG (SEQ ID NO: 6) 1586 NheI SEQ ID 3321-3340 GCT AGC GCG CAC (GCT AGC)NO: 2 TGC AAT GCC CTC (SEQ ID NO: 7)

To determine putative promoter activity, the reporter gene constructswere introduced into mammalian cells by transfection. In this study thecell line ND7/23 (ECACC Ref No: 92090903) was used. ND7/23 is a hybridcell line originating from a mouse neuroblastoma (N18tg2) fused by PEGto a rat dorsal root ganglion neuron cell line. This cell line waschosen since it express functional GABA_(B) receptors as evidenced byradioligand binding studies. Cells were cultured in a 1:1 mixture ofDulbecco's modified medium (DMEM) and Ham's F12 medium supplemented with10% (v/v) fetal bovine serum (FBS). Cells were grown at 37° C. in anatmosphere of 5% CO₂. ND7/23 cells (4×10⁵) were transfected using theDMRIE-C reagent according to manufacturer's protocol (Gibco). Briefly,cells were seeded in 6 well tissue culture plates (Nunc) the day beforetransfection. Next day, 2 μg of promoter-reporter construct and 1 μg oftransfection control construct (pSV-β-Galactosidase, Promega) was mixedwith 0.5 ml Optimem media (Gibco). DNA containing media was then mixedwith an equal volume (0.5 ml) of Optimem containing 4 μl of DMRIE-Creagent and the combined mixture was then incubated for 45 min. Afterincubation, the transfection mixture was added to the cells, which werewashed with Optimem media just prior to addition of transfectionmixture. Following 5 h incubation at 37° C., an equal volume of 1:1mixture of Dulbecco's modified medium (DMEM) and Ham's F12 mediumsupplemented with 20% (v/v) fetal bovine serum (FBS) was added to thecells. Cells were incubated for 24 h at 37° C. with or without cAMPenhancing supplement as indicated in FIG. 3. Before cell harvest, cellswere washed in PBS (7.6 mM Na₂HPO₄/NaH₂PO₄ pH 7.4 and 120 mM NaCl), thencell extracts were prepared by addition of 250 μl reporter lysis buffer(Promega) to cells, followed by transfer of cell suspension to 1.5 mltubes. Cells were further lysed by one round of freeze-thawing and 15 sof vortex. Cell debris were removed by 2 minutes of centrifugation at 12000 g. Luciferase activity in cell extracts was measured in a LuciferaseAssay System (Promega), where 40 μl of cell extracts was added to a96-well plate (Maxisorp, Nunc) and mixed with 50 μl of luciferasesubstrate. Luciferase activity was then measured in a LUMIstar (BMG Labtechnologies). As internal control for transfection efficiency,β-Galactosidase (β-Gal.) activity was measured in 96-well plate(Maxisorp, Nunc) using a β-Galactosidase Enzyme Assay (Promega)according to suppliers protocol. As seen in FIG. 3, transienttransfection of ND7/23 cells shows that both P1a and P1b has promoteractivity as pAM438, pAM436 and pAM440 (FIG. 3) result in reporterexpression, while the pAM442 (vector control) has very low activity. Inaddition, this experiment demonstrates that especially reporterexpression originating from pAM440 (R1b) can be induced by the cAMPactivating agent forskolin. Moreover, forskolin induced expression mayalso be further enhanced in the presence of the phosphodiesteraseinhibitor 1-methyl-3-isobutylxanthine (IBMX) although this experimentdoes not show a significant difference.

In conclusion, this experiment demonstrate that P1a and P1b both havepromoter activity and that the degree of activity can be modulated usingthe cAMP activating agent forskoline.

Example 3 Screening for Substances Modulating P1a and P1b Activity

Modulating GABA_(B)R1 expression in a controlled way is a means toregulate signaling through GABA_(B) receptors which could be ofsignificant therapeutic value for a variety of conditions. Particularly,the ability to specifically regulate expression of either 1a- or 1b-typeGABA_(B) receptor splice isoforms could be of medical value if theseisoforms could be attributed to specific conditions.

The experiment presented in Example 2 demonstrates that the use of P1aand P1b promoter/reporter constructions can be used to monitorGABA_(B)R1 expression in screens for therapeutic agents that can modifythe expression of GABA_(B) receptor 1 isoforms. A screen for P1a and P1bmodulating substances could be performed in ND7/23 cells as described inExample 2. A screen for P1a and P1b modulating substances could inaddition be performed in any cell type with endogenous expression ofGABA_(B) receptor 1 and 2 isoforms, in cells expressing recombinantGABA_(B) receptor 1 and 2 isoforms and in intact cells and in extract orfractions of cells expressing endogenous and recombinant proteinsmodulating GABA_(B) receptor function. Such screens could furthermore bedone in tissues and in living organisms.

Example 4 Functional Analysis of GABA_(B)-R1 Promoter Fragments andModified Forms

In order to identify functionally active promoter fragments of P1a andP1b a deletion analysis of DNA fragments containing promoter fragmentscan be performed in two steps.

It is anticipated that P1a and P1b comprise active fragments that canmediate increased expression by binding of transcription factors thatare activators as well as active fragments which can mediate decreasedexpression by binding of transcription factors that are repressors.

In the first step, promoter fragments mediating expression of a reportergene when used in reporter constructs can be stepwise deleted ortruncated to identify important regions. Briefly, truncated or deletedpromoter fragments are created by PCR using specific primers and thealready identified promoter sequences as template. Reporter constructs(as exemplified in this application) comprising the deleted or truncatedpromoter fragments are then created. These reporter constructs can thenbe used in transfection experiments, as described above, to identifyimportant regions of the promoters manifested in altered expression fromconstructs lacking active fragments compared to none-deleted constructs.

In the second step, the exact location and sequence of transcriptionfactor binding sites within active fragments can be determined by PCRtechnique using specific primers harbouring desired mutations. Suchpromoter fragments, with specifically mutated promoter regions, can beused in transfection experiments, similar to those described above, todetermine the exact sequence of functionally important nuclear factorbinding sites within active promoter fragments, manifested in alteredexpression from constructs with mutated DNA sequence compared tonone-mutated control constructs with equal size.

The above mentioned strategy can also be used to identify the specificactive promoter fragments which are important for the effect on promoteractivity of active substances identified when screening for therapeuticagents regulating GABA_(B)-R1 expression.

Example 5 Activity of GABA_(B)R1 Promoter P1a and P1b Fragments

Reporter constructs containing P1a and P1b promoter fragments weregenerated by PCR as described in Example 2 and fused to the fireflyluciferase reporter gene. The generated constructs are visualised inFIG. 4.

Putative transcription initiation sites were identified at position 3207in SEQ ID NO:1 for P1a and at position 4405 in SEQ ID NO:2 for P1b. Thepositions in the promoter region shown in FIG. 4 were calculated settingthe transcription initiation sites as position +1.

The generated promoter reporter constructs (see FIG. 4) were used totransfect ND7/23 cells. As shown in FIG. 4, deletion of P1a promoterregion in between position −2549 bp and −361 caused an increase inexpression, indicating putative repressor regions between position −2549and −361. Moreover, when 175 bp of the 5′ untranslated region wereremoved (compare the third and fourth P1a constructs from the top) therewas no detected difference in promoter activity and constructs with theshorter P1a 5′-untranslated region were therefore used for mutationalanalysis throughout the rest of the study. Additional deletions of theP1a promoter from position −361 to −46 caused a stepwise decrease of theexpression that seem to correlate with removal of promoter regionscontaining GC elements (I-III). This indicate that 361 bp of the P1apromoter can confer optimal expression and that promoter elementsessential for R1a-type expression, including GC elements I-III arelocated between position −361 and −46. Similar to the P1a promoter,deletion of P1b promoter region from position −3238 bp to −390 caused anincrease in expression, indicating putative repressor regions between−1084 and −390. Also, additional deletions of the P1b promoter fromposition −390 to −88 caused a stepwise decrease of the expression thatcorrelate with removal of promoter region GC elements (IV-VII) as wellas the consensus CRE. Although there was still some promoter activityleft when the P1b promoter region was deleted down to position −88, themajor promoter elements essential for R1b-type expression seem to belocated between position −390 and −88. Comparison of P1a and P1bexpression also indicates that, P1b mediated expression was higher(approximately 3-4 fold) than for P1a.

Example 6 Mutational Analysis of P1a and P1b Promoter Element Function

As shown in FIG. 4, reporter constructs −361 in P1a (short 5′UTR) and−390 in P1b conferred optimal expression of the respective promoters. Inorder to determine the importance of the promoter elements that wasfound within these regions, P1a (−361) and P1b (−390) constructs wereused as templates to obtain reporter constructs with promoter regionsmutated at specific sites. For the mutated promoter constructs, a 3 bpsubstitution was introduced by “quick change” site directed mutagenesis.

Site-directed mutagenesis: Mutant constructs were prepared with aQuickChange site-directed mutagenesis kit (Strategene, La Jolla, Calif.)according to the manufacturer's instructions. The mutagenicoligonucleotide primers are listed in Table 2. All constructs wereverified by DNA sequencing.

TABLE 2 Oligonucleotide primers used in the mutagenesis experimentsPrimer Primer Sequences 1949 P R1b Cre Fwd CGCCGCCCGTTTGGTCAGAGCCCCCT(SEQ ID NO: 8) 1950 P R1b Cre Rev AGGGGGCTCTGACCAAACGGGCGGCG (SEQ ID NO:9) 1951 P R1a GCI Fwd CTCTCTTCCCCCCTAACTGCCTTCCC (SEQ ID NO: 10) 1952 PR1a GCI Rev GGGAAGGCAGTTAGGGGGGAAGAGAG (SEQ ID NO: 11) 1953 P R1a GCIIFwd GGCGGTCCAGTTAGGGGCTGGGATCC (SEQ ID NO: 12) 1954 P R1a GCII RevGGATCCCAGCCCCTAACTGGACCGCC (SEQ ID NO: 13) 2051 P R1a GCIII FwdCCTCTCCACCGCCCTAACCACCGCGCTGTG (SEQ ID NO: 14) 2052 P R1a GCIII RevCACAGCGCGGTGGTTAGGGCGGTGGAGAGG (SEQ ID NO: 15) 2053 P R1b GCIVs FwdCCCCAGCTCCCGCCCTAACCCCCACCCC (SEQ ID NO: 16) 2054 P R1b GCIVs RevGGGGTGGGGGTTAGGGCGGGAGCTGGGG (SEQ ID NO: 17) 2055 P R1b GCV FwdCGCTTCCCTCCCCTAACCCTTCCTGCC (SEQ ID NO: 18) 2056 P R1b GCV RevGGCAGGAAGGGTTAGGGGAGGGAAGCG (SEQ ID NO: 19) 2057 P R1b GCVI FwdCCCTCCCCTCCCCTAACCTCCGACTGT (SEQ ID NO: 20) 2058 P R1b GCVI RevACAGTCGGAGGTTAGGGGAGGGGAGGG (SEQ ID NO: 21) 2059 P R1b GCVII FwdCTCCGCCCACCCCTAACTCCTGGCAC (SEQ ID NO: 22) 2060 P R1b GCVII RevGTGCCAGGAGTTAGGCGTGGGCGGAG (SEQ ID NO: 23) 2146 P R1b GCIVd FwdCCCCAGCTCCCTAACTAACCCCCACCCC (SEQ ID NO: 24) 2147 P R1b GCIVd RevGGGGTGGGGGTTAGTTAGGGAGCTGGGG (SEQ ID NO: 25)

Bases that are labelled bold correspond to designed mutations

Obtained promoter constructs were then used to transfect ND7/23 cells.As shown in FIG. 5, single point mutations of GC elements I, II and IIIreduced P1a expression to approximately 65-75% compared to wild-type,while double-mutations (I/II, I/III and II/III, respectively) resultedin a further decrease to approximately 55-60% of wild-type expressionlevels. When all three GC elements (I-III) were mutated, the reporterexpression was reduced to approximately one third (33%) of wild-typeexpression. Together, these results suggest that the three GC-elementsfound in P1a, all contribute to P1a mediated expression in a substantialand additive manner. However, the fact that one third of the P1apromoter activity still remains suggests that there are additionalpromoter elements within P1a contributing to P1a mediated expression.

In contrast to P1a, single mutations of the four P1b GC elements (IV, V,VI and VII) only caused small reductions of expression to approximately75-90% compared to wild-type. The relatively modest contribution by thefour P1b GC elements was also demonstrated by a promoter construct wereall four GC elements (IV-VII) had been mutated. This construct retained63% of P1b expression compared to the wild-type construct. However, whenthe P1b CRE consensus site was mutated (CRE), a dramatic reduction ofP1b mediated expression (approximately 50%) was obtained. Moreover, whenthe CRE mutation was combined with mutations of each GC elementrespectively, a further reduction was observed. Most notably, thepromoter construct containing a double mutation of the CRE and GC V(CRE/V) promoter elements resulted in a substantial reduction of P1bexpression to approximately 26%, similar to the construct where all fiveP1b promoter elements were mutated (CRE/IV-VII) where 24% of theexpression still remained compared to wild-type. Together these datademonstrates the absolute importance of the consensus CRE site for P1bexpression, alone or in combination with the GC elements (IV, V, VI andVII), of which GC element V seems to contribute most. The fact that onefourth of P1b promoter activity still remains suggests that, as for P1a,there are additional promoter elements within P1b that may contribute toP1b mediated expression.

Example 7 Identification of Factors Interacting with the P1b CRE Site

Electrophoretic Mobility Shift Assays (EMSA)

Electrophoretic mobility shift assays (EMSA) can be used to identifynuclear factors that interact with P1a and P1b promoter elements. In anattempt to identify factors interacting with the P1b CRE site, weperformed gelshift analysis with super-shift antibodies that recognisemembers of the CREB/ATF family of transcription factors. The DNA-bindingreactions (12 μl) were done as follows; 2-3 fmol ³²P-labeleddouble-stranded oligonucleotide corresponding to the P1b CRE site wasmixed with 5 μg crude nuclear extracts from ND7/23 cells and 1 μg poly(dI-dC), 25 mM Hepes (pH 7,9), 150 mM KCl, 5 mM dithiothreitol and 10%glycerol (Schneider et al 1986, Nucleic Acids Research. 14:1303-17). Inthe supershift lane (FIG. 6) the ATF-1 p35/CREB-1 p43/CREM-1 reactiveantibody (sc-270 from Santa Cruz Biotechnology, Santa Cruz, Calif.), waspre-incubated at room temperature for 20 min before addition of³²P-labeled probe. After incubation at room temperature for 15 min,protein-DNA complexes were resolved on precasted DNA-retard gels(Novex™) containing 6% polyacrylamide prepared with 0.5×TBE as gelbuffer. Following electrophoresis, gels were dried and visualised byautoradiography.

As shown in FIG. 6, addition of a monoclonal CREB/ATF supershiftantibody (reactive with members of the ATF/CREB family such as ATF-1p35, CREB-1 p43 and CREM-1 of mouse, rat and human origin) into the EMSAreaction mixture results in a distinct shift of the nuclear factor(s)that interacts with the P1b CRE site, while no super-shift was observedwhen the same antibody was added to other EMSA reactions containingother promoter elements (data not shown). This data suggests that thecomplex formed with the P1b CRE site contains a member of the CREB/ATFfamily.

Similar further studies can be performed in order to determine whichfactor(s) that interact with the various promoter elements in P1a andP1b.

Example 8 Use of Recombinant Transcription Factors in the Study of P1aand P1b Promoter Activities

Cells with stably integrated or with transiently transfected P1a/P1breporter constructs can be transfected with cDNA encoding specifictranscription factors to make reporter cells suitable for investigationsof P1a and P1b activities mediated by said transcription factors.Reporter cells can alternatively be generated by delivery oftranscription factors into similar cells by various means such as e.g.microinjection and lipofection. Reporter cells can be utilised forscreening of compounds, which are modulators of GABA_(B) receptor 1transcription.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. The human GABA_(B) receptor gene

The figure shows the organization of the human GABA_(B) receptor gene.Exons, represented by vertical squares/bars, are numbered (1-23).Translational start and stop sites are indicated by arrows. Location ofhuman GABA_(B) receptor 1 promoters P1a (SEQ ID NO: 1) and P1b (SEQ IDNO: 2) are indicated. The extent of human GABA_(B) receptor genomicsequence cloned in plasmid pAM 364 is indicated by a horizontal bar.

FIG. 2. Schematic representation of the P1a and P1b promoters

DNA fragments used to generate reporter constructs, corresponding to thepositions in the P1a and P1b promoter sequence, are shown below eachpromoter. Putative promoter elements in each promoter are indicated.Arrows indicate putative positions for start of transcription.

FIG. 3. Determination of GABA_(B) receptor 1 promoter P1a and P1bactivity

ND7/23 cells (4×10⁵) were transfected with promoter-luciferaseconstructs as described above. After transfection, cells were culturedin media without supplement (basal) or in the presence of Forskolin (10μM) or Forskolin (10 μM)+1-methyl-3-isobutylxanthine (0.125 mM) for 24h. After incubation, cells were harvested and luciferase activitymeasured. Luciferase activity, minus background, is shown as arbitrarylight units measured in 40 μl of cell extracts. Relative valuesrepresent the mean±SEM of two individual experiments.

FIG. 4: Deletion analysis of the GBR1 promoters in ND7/23 cells

ND7/23 cells (4×10⁵) were transfected with P1a and P1bpromoter-luciferase constructs as outlined above. After transfection,cells were cultured for 24 h, harvested and luciferase activity wasmeasured. Luciferase activity, minus background, is shown as arbitrarylight units measured in 10 μl of cell extracts. Relative valuesrepresent the mean±SEM of duplicate samples from at least threeindividual experiments.

FIG. 5. Effect of P1a and P1b promoter element mutations on promoteractivities.

ND7/23 cells (4×10⁵) were transfected with wild-type and mutated P1a andP1b promoter-luciferase constructs as described above. The pointmutations in the promoter constructs are outlined to the left. Aftertransfection, the cells were cultured for 24 h. After incubation, cellswere harvested and luciferase activity measured. Luciferase activity,minus background, is shown as arbitrary light units measured in 10 μl ofcell extracts. Relative values represent the mean±SEM of duplicatesamples from at least three individual experiments. Relative expressionlevel of mutated constructs are indicated to the left with the P1a andP1b “wild-type” expression level set as 100%.

FIG. 6. Identification of nuclear factors binding to the P1b consensusCRE site using CREB/ATF super-shift antibodies.

Nuclear extracts (5 μg) from ND7/23 cells were incubated withdouble-stranded ³²P-labeled oligonucleotides containing the P1bconsensus CRE site (sense:5′-CGCCGCCCGTGACGTCAGAGCCCCCT-3′ (SEQ ID NO:26)). In lane 1, no antibody was added. In lane 2, a mouse monoclonalantibody (sc-270 Santa Cruz Biotechnology, Santa Cruz, Calif.) reactivewith members of the ATF/CREB family such as ATF-1 p35, CREB-1 p43 andCREM-1 was pre-incubated at room temperature for 20 mm before additionof ³²P-labeled probe. The specific complex between nuclear factors andthe CRE is indicated by a star and the super-shifted complex isindicated by two stars.

1. An isolated nucleic acid molecule constituting a human GABA_(B)receptor 1 promoter P1a set forth as SEQ ID NO: 1, or a functionallyequivalent modified form thereof, or an active fragment thereof, whereinsaid modified form or active fraqment is at least 95% homologous to saidisolated molecule.
 2. The nucleic acid molecule according to claim 1selected from the group consisting of: (a) a nucleic acid moleculecomprising a nucleotide sequence set forth as SEQ ID NO: 1; and (b) anucleic acid molecule comprising a nucleotide sequence capable ofhybridizing to a filter-bound nucleotide sequence complementary to thepolypeptide codinq region of a DNA molecule as defined in (a) in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., withwashing in 0.1×SSC/0.1% SDS at 68° C.
 3. An isolated nucleic acidmolecule constituting a human GABA_(B) receptor 1 promoter P1b set forthas SEQ ID NO: 2, or a functionally equivalent modified form thereof, oran active fragment thereof, wherein said modified form or activefragment is at least 95% homologous to said isolated molecule.
 4. Thenucleic acid molecule according to claim 3 selected from the groupconsisting of: (a) a nucleic acid molecule comprising a nucleotidesequence set forth as SEQ ID NO: 2; and (b) a nucleic acid moleculecomprising a nucleotide sequence capable of hydridizing to afilter-bound nucleotide sequence complementary to the polypeptide codingregion of a DNA molecule as defined in (a) in 0.5 M NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., with washing in 0.1×SSC/0.1%SDS at 68° C.
 5. A nucleic acid molecule comprising a nucleic acidmolecule according to claim 1, in combination with a nucleic acidmolecule according to claim
 3. 6. A nucleic acid molecule comprising anucleic acid molecule according to claim 2, in combination with anucleic acid molecule accordinq to claim
 4. 7. A vector comprising anucleic acid molecule according to any one of claims 1 to
 4. 8. Anisolated cultured cell host comprising a vector according to claim
 7. 9.An isolated expression system comprising a nucleic acid moleculeconstituting a human GABA_(B) receptor 1 promoter P1a set forth as SEQID NO: 1, or a functionally equivalent modified form thereof, or anactive fragment thereof, wherein said modified form or active fragmentis at least 95% homologous to said isolated molecule.
 10. An expressionsystem according to claim 9, comprising a nucleic acid molecule selectedfrom the group consisting of: (a) a nucleicacid molecule comprising anucleotide sequence set forth as SEQ ID NO: 1; and (b) a nucleic acidmolecule comprising a nucleotide sequence capable of hybridizing to afilter-bound nucleotide sequence complementary to the polypeptide codingregion of a DNA molecule as defined in (a) in 0.5 H NaHPO₄, 7% sodiumdodecyl sulfate (SDS), 1 mM EDTA at 65° C., with washing in 0.1×SSC/0.1%SDS at 68° C.
 11. An isolated expression system comprising a nucleicacid molecule constituting a human GABA_(B) receptor 1 promoter P1b setforth as SEQ ID NO: 2, or a functionally equivalent modified formthereof, or an active fragment thereof, wherein said modified form oractive fragment is at least 95% homologous to said isolated molecule.12. An expression system according to claim 11, comprising a nucleicacid molecule selected from the group consisting of: (a) a nucleic acidmolecule comprising a nucleotide sequence set forth as SEQ ID NO: 2; and(b) a nucleic acid molecule comprising a nucleotide sequence capable ofhybridizing to a filter-bound nucleotide sequence complementary to thepolypeptide coding region of a DNA molecule as defined in (a) in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., withwashing in 0.1×SSC/0.1% SDS at 68° C.
 13. An isolated expression systemcomprising a nucleic acid molecule according to claim 1, in combinationwith a nucleic acid molecule according to claim
 3. 14. An isolatedexpression system comprising a nucleic acid molecule according to claim2, in combination with a nucleic acid molecule according to claim
 4. 15.An expression system according to any one of claims 9 to 12, which, inaddition, comprises a reporter gene.
 16. An expression system accordingto claim 15, wherein the reporter gene is selected from the groupconsisting of: (a) the firefly luciferase gene, (b) the bacterialchloramphenicol acetyl tranferase (CAT) gene, (c) the β-galactosidase(β-GAL) gene, and (d) the green fluorescent protein (GFP) gene.
 17. Anexpression system according to claim 15, wherein the promoter and thereporter gene are positioned so that the expression system of thereporter gene is regulated by the GABA_(B) receptor 1 promoter.
 18. Anexpression system according to any one of claims 9 to 12, wherein thenucleic acid molecule is present in a vector.
 19. An expression systemaccording to claim 18, wherein said vecor comprises an origin ofreplication and/or a dominant selection marker.
 20. An isolated hostcell transfected with an expression system according to ony one ofclaims 9 to
 12. 21. An expression system according to claim 16, whereinthe promoter and the reporter gene are positioned so that the expressionsystem of the reporter gene is regulated by the GABA_(B) receptor 1promoter.